With more than 170 million people infected worldwide, Hepatitis C Virus (HCV) has emerged as a significant public health burden. Unfortunately, there is no vaccine available to prevent this infection, and the only approved treatment has significant toxic side effects and is only effective in a subset of patients. Notably research efforts to understand HCV infection have been hindered by the lack of robust tissue culture infection systems and small animal models. Recently however, we and others have developed a robust in vitro HCV infection system, which should finally enable investigation of all stages of viral infection. In light of the immediate need to elucidate the biology of this virus and identify alternative HCV treatment option, the objective of this application is to develop our in vitro HCV infection system for high throughput screening (HTS) of potential antiviral compounds. The cell-based infection assay proposed would provide several advantages over the currently available HTS approaches being used for HCV drug discovery because it recapitulates the entire viral life cycle and does not restrict screening to any specific predetermined viral target(s). Thus, this HTS infection assay would have the potential to not only provide novel leads for drug discovery efforts, but also to identify new compounds with unique mechanisms of action that can be used as investigational tools to study HCV infection. Accordingly, this study has 3 Specific Aims: 1) Implement, optimize, and validate a FRET-based NS3 protease HCV infection assay amenable to HTS. 2) Implement, optimize, and validate secondary screens that can be used to prioritize initial hits based on specificity, efficacy, and toxicity. 3) Verify the high throughput capacity of the system by performing preliminary, proof-of-principle screens and analyzing hits to target and/or mechanism of action.